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is displayed in Figure
2
. A list of automated labware
position (ALP) hardware is presented in Table
1
. The
method employs individual Span-8 probes to deliver
enzyme and reagent transfers while DNA cleanup, wash,
and elution transfers are performed using the multichannel
96 pipetting head. A static Peltier unit ensures that enzyme
master mixes are kept cool during the course of the
method. For on-deck incubations, a Biometra T-Robot
integration is required, otherwise the method prompts
the user to remove the plate from the deck and perform
the incubation in an off-deck thermocycler. Filter tips are
employed throughout the method to reduce the possibility
of instrument or sample contamination. A list of automation
consumables and user-supplied reagents can be found
in Tables 2 and 3, respectively.
The automation method follows NEBNext Ultra DNA
Library Preparation Kit for Illumina protocol with a few
modifications. Sheared gDNA is added to a 96-well plate
by the user, along with the master mixes and reagents
as directed by the reagent calculator. The NEBNext
adapter is kept separate from the Ligation Master Mix
to inhibit the formation of adapter dimers prior to the
adapter ligation step. Another important modification
to the NEBNext Ultra DNA Library Preparation Kit for
Illumina protocol involves the USER enzyme. The USER
enzyme has been integrated into the PCR
1
master mix,
requiring a
1
5-minute, 37°C incubation added to the
beginning of the PCR program outlined in the NEBNext
Ultra DNA Library Preparation Kit for Illumina protocol.
This reduces the number of tips required by the protocol
in addition to offering time savings overall. Lastly, the
automation method employs Beckman Coulter’s SPRIselect
reagent in place of AMPure XP to provide for more
consistent size selection. The automation workflow is
presented in Figure
1
.
The automated NEBNext DNA Ultra method utilizes
an HTML-driven User Interface (UI) that allows the user
to customize their workflow by offering a number of
different options. Some major options offered by the
UI include the following:
1
.
Selecting any number of samples to process,
between
1
and 96.
2.
Selecting which procedures of the library
construction workflow to run.
3.
Selecting from an extensive list of size selection
options to be used in the library construction
procedure (
1
50 bp, 200 bp, 250 bp, 300–400 bp,
400–500 bp, 500–700 bp inser ts or no size
selection at all).
4.
Choosing to conduct on-deck or off-deck incubations.
5.
User-defined transfer volumes for various steps to
further increase the flexibility of the method.
An image of the user interface is presented in Figure 3.
Fig. 3.
NEBNext Ultra DNA user interface.
1.
2.
3.
4.
5.
Fig. 2.
NEBNext Ultra DNA automated deck layout with integrated
Biometra T-Robot.
AAG-350TCH08.14-A